**1. Introduction**

Stool suspension is commonly used as a straightforward, cheap and non-invasive material for treating several conditions and diseases. Outcomes of fecal microbiota transplantation (FMT) are encouraging in many diseases (e.g., inflammatory bowel diseases [1]) and excellent in others (e.g., *Clostridioides di*ffi*cile* infection, especially as a life-saving therapy [2,3]). Not surprisingly, there are more than 250 FMT clinical trials completed or ongoing worldwide [4].

Despite the thriving of the FMT trials, our knowledge on the real composition of "healthy microbiota" is still scarce. We do not exactly know how the transplanted bacteria survive, colonize, and function in the recipient's gu<sup>t</sup> or, most importantly, which methods may, or should, be used to diagnose and monitor transplanted feces to assess whether the fecal microbiota solution is appropriate for transplantation and consists of a "healthy microbiota". We also acknowledge that a healthy gu<sup>t</sup> microbiota requires proper virus and fungi composition [5,6].

Analyses of human intestinal microorganisms were, until recently, mostly performed by culture-dependent methodologies, limiting the screened biodiversity only to the cultivable species, although it is known that only about 15–20% of microbes living in the human gu<sup>t</sup> are cultivable as of now. The availability of novel tools, primarily next-generation sequencing (NGS), has enabled the assessment of marker taxonomical genes and even whole genomes retrieved from the complex microbial communities. The most widely used NGS method for the taxonomic and phylogenetic evaluation of bacterial community composition relies on 16S rRNA gene PCR amplicon analysis [7–9].

Currently, it can be observed that the market o ffering fecal microbiota suspensions and tools for FMT suspensions assessments is growing rapidly. Companies provide material from di fferent donors, using various methodologies for testing the donors and stool processing. However, each of these methods is usually applied separately, and on these distinct analyses far-reaching conclusions are built. There are very few reports comparing di fferent methods for the assessment of gu<sup>t</sup> microbiota [10–13] and indicating how to assess the stability of fecal microbiota in the donors. There is also a need to identify "super-donor" units, i.e., persons whose microbiota contains all relevant microbes and potentially can cure the vast majority of microbiota-related diseases and conditions [14].

There is also a need for defining "super-donors" for FMT. It is postulated that there are individuals whose gu<sup>t</sup> microbiome possesses certain characteristics, such as the presence or absence of specific (unfortunately not fully recognized) bacteria, phages and metabolites, which protect the donor from the vast majority of gut-related dysbiosis [14]. Furthermore, it is postulated that these individuals are the most desired donors (namely "super-donors"), and that their fecal microbiota is highly suitable for transplantation. Theoretically, such donors should be adequate for every FMT intervention. However, recent studies prove that there are some (still unknown) specific features making these (super) donors not so universal [15,16]. Therefore, an algorithm for finding perfectly matching donor–recipient couples still needs elucidation. We believe that this coupling of donor and recipient may be possible, but this requires more data and more diagnostic/analytical tools. To define what exactly "super-donor" means, we need to have more well-designed comparable clinical trials for dysbiosis-related diseases [17]. Only then will we be able to conduct a general investigation, looking for features common to all donors. Our study is in line with this general quest in medicine. Summarizing, for the purpose of this study, the term "super-donor" was used with its complex definition, including an indication that the microbiota of the "super-donor" has all the necessary beneficial components to maintain human welfare and that it will most probably cure the majority of dysbiosis. Besides, the most important conclusion must be the statement that the donor can be defined as a "super-donor" only when the majority (ideally all) clinical outcomes of FMT (from this donor) are good.

In this study, we conducted a series of experiments using a multi-method approach to trace the stability of the composition of the gu<sup>t</sup> microbiota in various donors over time and to find the most suitable method for assessing the quality of the gu<sup>t</sup> microbiota for the proper selection of fecal donors and to pave the way to find "super-donors". Moreover, we were looking for bacterial indicators of "good" and/or "bad" donors to simplify and parametrize the selection of suitable givers of stool samples for FMTs. We hypothesized that various methodologies of microbiota assessment to evaluate donors gives more data than each method separately.

#### **2. Material and Methods**

### *2.1. Stool Donors*

Ten consecutive stools donated by each of three donors were used for the experiments (30 stools in total). The donors of stool were randomly selected males (donors named A and B) and an intentionally chosen male (donor C, that is, a regular stool donor registered in the Polish stool bank). They were selected according to our protocol published previously [18]. Donor C was selected with respect to criteria described by Cammarota et al. [19]. All donors were screened by a questionnaire based on international guidelines that was presented in our previous publication [18]. Briefly, one of them (donor C; male, 28 years old, healthy, with normal BMI) was a regular donor of feces, for the purpose of producing a preparation for fecal microbiota transplantation (chosen from the stool donor bank) and the other two were randomly selected males (donor A—male, 16 years old with food allergy, recurrent aphthous stomatitis and normal BMI, and donor B—male, 55 years old, a medical worker with inhaled allergy and a BMI of 27). A medical questionnaire with basic data was received from each person. Each feces sample was prepared in the same time frame and in the same way by homogenizing, diluting in normal saline, and sieving through sterile gauze or sieves to obtain a clear, homogeneous fluid being a suspension of feces. This is the regular way of producing feces for use as FMT [18]). The material prepared in this way was divided into three parts—one for assessment by flow cytometry in the LIVE/DEAD method (Molecular Probes, Eugene, OR, USA), the other for performing classical culturing, and the third for immediate isolation of DNA for V3V4 16S rDNA variable region sequencing (90 samples in total).
